The instant invention is directed to compounds useful for inhibiting protein tyrosine phosphatase PTP1B, preparation of the compounds, compositions containing the compounds, and treatment of diseases using the compounds.
PTP1B belongs to a family of protein tyrosine phosphatases involved in the regulation of the cellular signaling mechanisms which are involved in metabolism, growth, proliferation, and differentiation (Science 253:401-6 (1991)). Overexpression or altered activity of tyrosine phosphatase PTP1B can contribute to the progression of various diseases (Ann. Rev. Biochem., 54:897-930 (1985)); and there is evidence which suggests inhibition of protein tyrosine phosphatase PTP1B is therapeutically beneficial for the treatment of diseases such as type I and II diabetes, obesity, autoimmune disease, acute and chronic inflammation, osteoporosis, and various forms of cancer (J. Natl. Cancer Inst. 86:372-8 (1994); Mol. Cell. Biol. 14: 6674-6682 (1994); The EMBO J. 12:1937-46 (1993); J. Biol. Chem. 269:30659-30667 (1994); and Biochemical Pharmacology 54:703-711 (1997)).
Because of the important role played by unregulated protein tyrosine phosphatase PTP1B in these diseases, agents which inhibit the enzyme have been the subject of active current research for their clinical potential. Reference is made to WO 99/46236, WO 99/46237, WO 99/46267 and WO 99/46268; and although each teaches protein tyrosine phosphatase PTP1B inhibitors, there is still a need for protein tyrosine phosphatase PTP1B inhibitors with modified or improved profiles of activity.
In its principle embodiment, therefore, the instant invention provides compounds of formula (I) 
or therapeutically acceptable salts thereof, wherein
R1 is selected from the group consisting of benzodioxolyl, dibenzofuranyl, indolyl, phenyl, and thianthrenyl;
wherein the benzodioxolyl, the dibenzofuranyl, the indolyl, and the thianthrenyl can be optionally substituted with one, two, three, or four substituents independently selected from the group consisting of alkanoyl, alkoxy, alkoxycarbonyl, alkyl, amino, aryl, arylalkyl, carbonyloxy, carboxy, cyano, cycloalkyl, cycloalkylalkyl, halo, hydroxy, hydroxyalkyl, nitro, perfluoroalkoxy, perfluoroalkyl, and thioalkoxy; and
wherein the phenyl is substituted with one, two, three, or four substituents independently selected from the group consisting of perfluoroalkoxy and phenyl.
In still another embodiment the instant invention provides a method for inhibiting protein tyrosine phosphatase comprising administering a therapeutically effective amount of a compound of formula (I).
In still another embodiment the instant invention provides a method for treating diseases in a patient in recognized need of such treatment comprising administering to the patient a therapeutically effective amount of a compound of formula (I).
In still another embodiment the instant invention provides a composition comprising a compound of formula (I) in combination with a therapeutically acceptable excipient.
The instant invention provides a series of compounds which inhibit protein tyrosine phosphatase PTP1B. As used throughout the specification of the instant invention, the following terms, as used herein, have the meanings indicated:
The term xe2x80x9calkanoyl,xe2x80x9d as used herein, represents an alkyl group attached to the parent molecular moiety through a carbonyl group.
The term xe2x80x9calkoxy,xe2x80x9d as used herein, represents an alkyl group attached to the parent molecular moiety through an oxygen atom.
The term xe2x80x9calkoxycarbonyl,xe2x80x9d as used herein, represents an alkoxy group attached to the parent molecular moiety through a carbonyl group.
The term xe2x80x9calkyl,xe2x80x9d as used herein, represents a saturated, monovalent straight or branched chain hydrocarbon having from one to six carbons.
The term xe2x80x9camino,xe2x80x9d as used herein, represents xe2x80x94NR2R3, wherein R2 and R3 are independently selected from the group consisting of hydrogen, alkanoyl, alkoxycarbonyl, alkyl, cycloalkyl, cycloalkylalkyl, a nitrogen protecting group, phenyl, and phenylalkyl; or R2 and R3, together with the nitrogen atom to which they are attached, form a ring selected from the group consisting of morpholinyl, oxazinanyl, piperazinyl, piperidinyl, and pyrrolidinyl.
The term xe2x80x9caryl,xe2x80x9d as used herein, represents dihydronaphthyl, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. Aryl groups having an unsaturated or partially saturated ring fused to an aromatic ring can be attached through the saturated or the unsaturated part of the group.
The term xe2x80x9carylalkyl,xe2x80x9d as used herein, represents an aryl group attached to the parent molecular moiety through an alkyl group.
The term xe2x80x9ccarbonyl,xe2x80x9d as used herein, represents xe2x80x94C(O)xe2x80x94.
The term xe2x80x9ccarbonyloxy,xe2x80x9d as used herein, represents an alkanoyl group attached to the parent molecular group through an oxygen atom.
The term xe2x80x9ccarboxy,xe2x80x9d as used herein, represents xe2x80x94CO2H.
The term xe2x80x9ccyano,xe2x80x9d as used herein, represents xe2x80x94CN.
The term xe2x80x9ccycloalkyl,xe2x80x9d as used herein, represents a monovalent saturated cyclic or bicyclic hydrocarbon group of three to twelve carbons.
The term xe2x80x9ccycloalkylalkyl,xe2x80x9d as used herein, represents a cycloalkyl group attached to the parent molecular moiety through an alkyl group.
The term xe2x80x9chalo,xe2x80x9d as used herein, represents F, Cl, Br, or I.
The term xe2x80x9chydroxy,xe2x80x9d as used herein, represents xe2x80x94OH.
The term xe2x80x9chydroxyalkyl,xe2x80x9d as used herein, represents a hydroxy group attached to the parent molecular group through an alkyl group.
The term xe2x80x9cnitro,xe2x80x9d as used herein, represents xe2x80x94NO2.
The term xe2x80x9cnitrogen protecting group,xe2x80x9d as used herein, represents selectively introducible and removable groups which protect amino groups against undesirable side reactions during synthetic procedures. Examples of amino protecting groups include methoxycarbonyl, ethoxycarbonyl, trichloroethoxycarbonyl, benzyloxycarbonyl (Cbz), chloroacetyl, trifluoroacetyl, phenylacetyl, formyl, acetyl, benzoyl, tert-butoxycarbonyl (Boc), para-methoxybenzyloxycarbonyl, isopropoxycarbonyl, phthaloyl, succinyl, benzyl, diphenylmethyl, triphenylmethyl (trityl), methylsulfonyl, phenylsulfonyl, para-toluenesulfonyl, trimethylsilyl, triethylsilyl, triphenylsilyl, and the like.
The term xe2x80x9cperfluoroalkoxy,xe2x80x9d as used herein, represents a perfluoroalkyl group attached to the parent molecular moiety through an oxygen atom.
The term xe2x80x9cperfluoralkyl,xe2x80x9d as used herein, represents an alkyl group in which all of the hydrogen atoms have been replaced with fluoride atoms.
The term xe2x80x9cphenylalkyl,xe2x80x9d as used herein, represents a phenyl group attached to the parent molecular group through an alkyl group.
The term xe2x80x9cthioalkoxy,xe2x80x9d as used herein, represents an alkyl group attached to the parent molecular moiety through a sulfur atom.
The instant compounds can exist as therapeutically acceptable salts. The term xe2x80x9ctherapeutically acceptable salt,xe2x80x9d refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible; suitable for treatment of diseases without undue toxicity, irritation, and allergic response; commensurate with a reasonable benefit/risk ratio; and effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the amino group of the compounds with a suitable acid. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetic, trifluoroacetic, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric, and the like. The amino groups of the compounds can also be quaternized with alkyl chlorides, bromides, and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl, and the like.
Basic addition salts can be prepared during the final isolation and purification of the instant compounds by reaction the carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributlyamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,Nxe2x80x2-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like, are contemplated as being within the scope of the instant invention.
The instant compounds can also exist as therapeutically acceptable prodrugs. The term xe2x80x9ctherapeutically acceptable prodrug,xe2x80x9d refers to those prodrugs or zwitterions which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
The term xe2x80x9cprodrug,xe2x80x9d refers to compounds which are rapidly transformed in vivo to parent compounds of formulas (I) and (II) for example, by hydrolysis in blood.
Asymmetric centers can exist in the instant compounds. Individual stereoisomers of the compounds are prepared by synthesis from chiral starting materials or by preparation of racemic mixtures and separation by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, or direct separation of the enantiomers on chiral chromatographic columns. Starting materials of particular stereochemistry are either commercially available or are made by the methods described hereinbelow and resolved by techniques well-known in the art.
Therapeutic compositions of the instant compounds comprise an effective amount of the same formulated with one or more therapeutically acceptable excipients. The term xe2x80x9ctherapeutically acceptable excipient,xe2x80x9d refers to a non-toxic, solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Examples of therapeutically acceptable excipients include sugars; cellulose and derivatives thereof; oils; glycols; solutions; buffering, coloring, releasing, coating, sweetening, flavoring, and perfuming agents; and the like. These therapeutic compositions can be administered parenterally, intracisternally, orally, rectally, or intraperitoneally.
Liquid dosage forms for oral administration of the instant compounds comprise formulations of the same as emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the compounds, the liquid dosage forms can contain diluents and/or solubilizing or emulsifying agents. Besides inert diluents, the oral compositions can include wetting, emulsifying, sweetening, flavoring, and perfuming agents.
Injectable preparations of the instant compounds comprise sterile, injectable, aqueous and oleaginous solutions, suspensions, or emulsions, any of which can be optionally formulated with parenterally acceptable diluents, dispersing, wetting, or suspending agents. These injectable preparations can be sterilized by filtration through a bacterial-retaining filter or formulated with sterilizing agents which dissolve or disperse in the injectable media.
PTP inhibition by the instant compounds can be delayed by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compounds depends upon their rate of dissolution which, in turn, depends on their crystallinity. Delayed absorption of a parenterally administered compound can be accomplished by dissolving or suspending the compound in oil. Injectable depot forms of the compounds can also be prepared by microencapsulating the same in biodegradable polymers. Depending upon the ratio of compound to polymer and the nature of the polymer employed, the rate of release can be controlled. Depot injectable formulations are also prepared by entrapping the compounds in liposomes or microemulsions which are compatible with body tissues.
Solid dosage forms for oral administration of the instant compounds include capsules, tablets, pills, powders, and granules. In such forms, the compound is mixed with at least one inert, therapeutically acceptable excipient such as a carrier, filler, extender, disintegrating agent, solution retarding agent, wetting agent, absorbent, or lubricant. With capsules, tablets, and pills, the excipient can also contain buffering agents. Suppositories for rectal administration can be prepared by mixing the compounds with a suitable non-irritating excipient which is solid at ordinary temperature but fluid in the rectum.
The instant compounds can be micro-encapsulated with one or more of the excipients discussed previously. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric and release-controlling. In these forms, the compounds can be mixed with at least one inert diluent and can optionally comprise tableting lubricants and aids. Capsules can also optionally contain opacifying agents which delay release of the compounds in a desired part of the intestinal tract.
Transdermal patches have the added advantage of providing controlled delivery of the instant compounds to the body. Such dosage forms are prepared by dissolving or dispensing the compounds in the proper medium. Absorption enhancers can also be used to increase the flux of the compounds across the skin, and the rate of absorption can be controlled by providing a rate controlling membrane or by dispersing the compounds in a polymer matrix or gel.
Diseases caused or exacerbated by protein tyrosine phosphatase PTP 1B activity are treated or prevented in a patient by administering to the same a therapeutically effective amount of the instant compounds in such an amount and for such time as is necessary to achieve the desired result. The term xe2x80x9ctherapeutically effective amount,xe2x80x9d refers to a sufficient amount of the compound to treat protein tyrosine phosphatase PTP1B activity at a reasonable benefit/risk ratio applicable to any medical treatment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, rate of excretion; the duration of the treatment; and drugs used in combination or coincidental therapy.
The total daily dose of the instant compounds in single or divided doses can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions can contain such amounts or submultiples thereof of the compounds to make up the daily dose. In general, treatment regimens comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compounds per day in single or multiple doses.
Specific compounds of the invention include, but are not limited to, (2R)-2-(2,6-dichloro-4-dibenzo(b,d)furan-4-ylphenoxy)-3-phenylpropanoic acid; (2R)-2-((3,5-dichloro-4xe2x80x2-(trifluoromethoxy)(1,1xe2x80x2-biphenyl)-4-yl)oxy)-3-phenylpropanoic acid; (2R)-2-((3,5-dichloro-3xe2x80x2-phenyl(1,1xe2x80x2-biphenyl)-4-yl)oxy)-3-phenylpropanoic acid; (2R)-2-(2,6-dichloro-4-(1H-indol-5-yl)phenoxy)-3-phenylpropanoic acid; (2R)-2-(2,6-dichloro-4-(1-thianthrenyl)phenoxy)-3-phenylpropanoic acid; and (2R)-2-(4-(1,3-benzodioxol-5-yl)-2,6-dichlorophenoxy)-3-phenylpropanoic acid.
Determination of Biological Activity
Purification of Human Protein Tyrosine Phosphatase 1B from E. coli 
Human protein tyrosine phosphatase 1B (PTP1B, amino acid residues 1-321) was expressed in E. coli BL21(DE3). The cell paste was resuspended in 4 cell paste volumes of lysis buffer containing 100 mM MES (pH 6.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 20 U/mL Benzonase, 0.5 mg/mL lysozyme, and 1 mM MgCl2 and incubated for 35 minutes at room temperature. The cells were lysed at 11,000 psi using a Rannie homogenizer, and the homogenate was clarified in a Beckman GSA rotor at 10,000xc3x97g for 30 minutes at 4xc2x0 C. The supernatant was loaded onto a 5xc3x9721 cm S-Sepharose-FF column (Amersham Pharmacia Biotech) pre-equilibrated with 5 column volumes of buffer containing 100 mM MES (pH 6.5), 100 mM NaCl, 1 mM EDTA, and 1 mM DTT and eluted with 10 column volumes of the same. The fractions (28 mL each) were assayed for protein by 10-20% Tris-Glycine SDS-PAGE. Fractions which contained  greater than 95% protein tyrosine phosphatase 1B were combined.
Protein Tyrosine Phosphatase 1B Activity Assay
Protein tyrosine phosphatase 1B activity was determined by measuring the phosphate release from triphosphorylated peptide which corresponds to residues 1135-1156 of the xcex2-subunit of the human insulin receptor (xcex2IRK substrate) as described in Nature, 1985, 313, 756-761. Protein tyrosine phosphatase 1B activity was determined in a final assay volume of 50 xcexcL containing 50 mM Tris HCl, 50 mM Tris Base, 150 mM NaCl, 3 mM DTT, 2 nM protein tyrosine phosphatase 1B(1-321), and 20 xcexcM xcex2IRK substrate. Various concentrations of test compounds in 5 xcexcL of 10% DMSO were incubated for 5 minutes at room temperature in assay buffer (25 xcexcl) containing 20 xcexcM xcex2IRK substrate in a round-bottom microtiter plate (Costar) pre-coated with 1% bovine serum albumin. The assay was initiated by the addition of protein tyrosine phosphatase 1B enzyme (20 xcexcl)in assay buffer. After 10 minutes of incubation at room temperature, the reaction was terminated by the addition of 100 xcexcL of malachite green (Upstate Biotechnology Inc.) containing 0.01% Tween-20. After a 5 minute incubation, quantitation of free phosphate released from the xcex2IRK substrate was determined in a Victor II plate reader (Wallac; Turku, Finland) by measuring the absorbence of the malachite green at 620 nm.
The instant compounds were found to inhibit protein tyrosine phosphatase 1B with inhibitory potencies under 35 xcexcM. As protein tyrosine phosphatase 1B inhibitors, therefore, the instant compounds are useful for treating diseases caused by overexpressed or altered protein tyrosine phosphatase 1B activity. These diseases include autoimmune diseases, acute and chronic inflammatory diseases, osteoporosis, obesity, cancer, malignant diseases, and type I and type II diabetes.
Synthetic Methods
Abbreviations which have been used in the descriptions of the scheme and the examples that follow are: DEAD for diethyl azodicarboxylate; DIAD for diisopropyl azodicarboxylate; THF for tetrahydrofuran; dba for dibenzylideneacetone; and DMSO for dimethylsulfoxide.
The compounds and processes of the instant invention will be better understood in connection with the following synthetic scheme which illustrates the methods by which the compounds of the invention may be prepared. The group R1 is as defined above unless otherwise noted below. 
As shown in Scheme 1, compounds of formula (2) can be reacted with 4-bromo-2,6-dichlorophenol (3) in the presence of a trialkylphosphine or triarylphosphine and a diazo compound to provide compounds of formula (4). Representative trialkylphosphines include tributylphosphine and trimethylphosphine; representative triarylphosphines include triphenylphosphine and tri-o-tolylphosphine; and representative diazo compounds include DEAD and DIAD. Solvents commonly used in these reactions include THF, diethyl ether, and methyl tert-butyl ether. The reaction is conducted at about 20xc2x0 C. to about 40xc2x0 C., and typical reaction times are about 30 minutes to about 12 hours.
Compounds of formula (4) can be coupled to compounds of formula (5) in the presence of a palladium catalyst and base to provide compounds of formula (6). Representative palladium catalysts include Pd(PPh3)4, PdCl2(PPh3)2, and Pd2(dba)3 with PPh3. Examples of bases include CsF, K2CO3, Na2CO3, and Cs2CO3. Solvents commonly used in these reactions include toluene, benzene, and xylene. The reaction temperature about 80xc2x0 C. to about 115xc2x0 C., and depends on the solvent and reagents chosen. Reaction times are typically about 12 to about 24 hours.
Conversion of compounds of formula (6) to compounds of formula (I) can be accomplished under hydrolysis conditions. Representative hydrolyzing reagents include LiOH, KOH, and NaOH. Examples of solvents used in these reactions include methanol, water, tetrahydrofuran, and mixtures thereof. The reaction is conducted at about 20xc2x0 C. to about 40xc2x0 C., and reaction times are typically about 1 to about 6 hours.